1. Field
This invention relates to manufacturing scale mammalian cell culture technology and specifically to a new seed-train expansion method for mammalian cells. Cryopreserved cells are used to inoculate directly into a newly designed bioreactor, which serves as a seed source for production scale.
2. Background
The start of a new cell seed-train expansion for a production campaign for the expression of a mammalian cell culture system is a critical process step. Operational inconsistencies and errors often compromise the process, leading to significant delays and inoculum variability. In typical production protocols, a new seed train expansion begins from a 1-2 mL cryovial (master working cell bank, MWCB). The cell concentration of this container is usually in the range of 5-10 million cells/mL. The cells are thawed, washed to remove the cryoprotectant and then first cultured (seeded) in small tissue culture flasks. It is standard practice with mammalian cell cultures to use inoculation densities of 0.5 to 1×106 cells/mL. Seeding cells at lower cell concentrations after cryopreservation may result in an extended lag phase prior to entering the growth phase, poor cell performance or even cell death. This is especially accentuated in the serum-free or even protein-free cell culture media that has become the modus operandi for current cell culture manufacturing.
The cells are sub-cultivated according to their specific growth rate (cell density), and are usually split into multiple cell culture flasks every 2-3 days. Once enough biomass is produced, the cells are expanded into larger cultivation bottles such as roller bottles, shake or spinner flasks. After enough cell mass is accumulated, a bioreactor, which becomes the seed culture for the production-scale vessel, is inoculated. This described seed-train expansion is general practice for several mammalian cell lines and is widely used in commercial production and academia. An overview of a commercial seed train expansion using T-flasks and spinner flasks is also given by Whitaker et al., Journal of the American Chemical Society, pages 28-43, 1998.
Typical scale-up protocols which take about four to six weeks to complete under optimal conditions are labor intensive and are susceptible to contamination and variability. The small scale conditions are not well defined—usually there is no set-point for pH and DO during the T-flask, roller bottle or shaker flask period, which may lead to more variability throughout the process and might compromise the quality of the inoculum and the product.